Pharmaceutical compositions

ABSTRACT

An oral or injectable pharmaceutical composition is provided for treating diseases caused by retroviruses or hepatitis B viruses. The composition comprises a therapeutically effective amount of at least one anti-retroviral drug and a therapeutically effective amount of at least one pharmacokinetic booster or enhancer or derivative thereof. Methods and kits are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to co-pending Indian Provisional PatentApplication Serial Number 201621005051, filed on Feb. 12, 2016,co-pending Indian Provisional Patent Application Serial Number201621032504, filed on Sep. 23, 2016, and co-pending Indian ProvisionalPatent Application Serial Number 201621040945, filed on Nov. 30, 2016.These applications are incorporated herein by reference, in theirentireties.

FIELD

The present invention relates to pharmaceutical compositions comprisingat least one anti-retroviral drug and at least one pharmacokineticbooster or enhancer. The present invention also provides themanufacturing process thereof and use of the said compositions for theprevention, treatment or prophylaxis of diseases caused by virusesspecifically caused by retroviruses or hepatitis B virus.

BACKGROUND

Human Immunodeficiency Virus (HIV), the virus that causes AcquiredImmune Deficiency Syndrome (AIDS) has become one of the world's mostserious health concern. HIV belongs to a class of viruses calledretroviruses. Retroviruses are RNA (ribonucleic acid) viruses, and toreplicate (duplicate), the viruses must make a DNA (deoxyribonucleicacid) copy of their RNA. It is the DNA genes that allow the virus toreplicate. Like all viruses, HIV can replicate only inside cells,commandeering the cell's machinery to reproduce. Only HIV and otherretroviruses, however, once inside a cell, use an enzyme called reversetranscriptase to convert their RNA into DNA, which can be incorporatedinto the host cell's genes.

HIV destroys CD₄ positive (CD₄) T cells, which are white blood cellscrucial to maintaining the function of the human immune system. Thedestruction of these cells leaves people infected with HIV vulnerable toother infections, diseases and other complications. These cells,sometimes called “T-helper cells,” play a central role in the immuneresponse, signalling other cells in the immune system to perform theirspecial functions. As HIV attacks these cells, the person infected withthe virus is less equipped to fight off infection and disease,ultimately resulting in the development of AIDS.

A healthy, uninfected person usually has 800 to 1,200 CD₄ T cells percubic millimeter (mm³) of blood. HIV appears to have a particularaffinity for the human T-4 lymphocyte cell which plays a vital role inthe body's immune system. HIV infected white blood cells (WBCs) lead toa decrease in WBC population. Eventually, the immune system is renderedinoperative and ineffective against various opportunistic diseases.During untreated HIV infection, the number of these cells in a person'sblood progressively declines. When the CD₄ T cell count falls below200/mm³, a person becomes particularly vulnerable to the opportunisticinfections and cancers that typify AIDS, the end stage of HIV disease.People with AIDS often suffer infections of the lungs, intestinal tract,brain, eyes, and other organs, as well as debilitating weight loss,diarrhea, neurologic conditions, and cancers such as Kaposi's sarcomaand certain types of lymphomas.

The first case was reported in 1981 and today there are approximately36.9 million people currently living with HIV and tens of millions ofpeople have died of AIDS-related causes since the beginning of theepidemic. While new cases have been reported in all regions of theworld, approximately 70% are in sub-Saharan Africa. Further, as per the2016 fact sheet of UNAIDS, in 2015, there were 36.7 million peopleliving with HIV. As of December 2015, 17 million people living with HIVwere accessing antiretroviral therapy. In 2015, 1.1 million people diedfrom AIDS-related causes worldwide.

HIV is the causative agent of AIDS that has created a major health careproblem not only in India but globally. AIDS causes a gradual breakdownof the body's immune system as well as progressive deterioration of thecentral and peripheral nervous systems. Since its initial recognition inthe early 1980s, AIDS has spread rapidly and has now reached epidemicproportions within a relatively limited segment of the population.Intensive research has led to the discovery of the responsible agent,human T-lymphotropic retrovirus 111 (HTLV-111) commonly referred to asthe Human Immunodeficiency Virus or HIV.

Currently available antiretroviral drugs for the treatment of HIVinclude: zidovudine or AZT (Retrovir®), didanosine or DDI (Videx®),stavudine or D4T (Zenith®), lamivudine or 3TC (Epivir®), zalcitabine orDDC (Hivid®), abacavir sulphate (Ziagen®), tenofovir disoproxil fumarate(Viread®), emtricitabine (Emtriva®), Combivir® (contains 3TC and AZT),Trizivir® (contains abacavir, 3TC and AZT), Epzicom® (contains abacavirand lamivudine); nevirapine (Viramune®), delavirdine (Rescriptor®),efavirenz (Sustiva®), saquinavir (Invirase®, Fortovase®), indinavir(Crixivan®), ritonavir (Norvir®), nelfinavir (Viracept®), amprenavir(Agenerase®), atazanavir (Reyataz®), Evotaz® (contains atazanavir andcobicistat), fosamprenavir (Lexiva®), Kaletra® (contains lopinavir andritonavir), enfuvirtide (T-20, Fuzeon®), Truvada® (contains Tenofovirand Emtricitabine), darunavir (Prezista®), Prezcobix® (containsdarunavir and cobicistat), dolutegravir (Tivicay®), Triumeq® (containsdolutegravir, abacavir and lamivudine), elvitegravir (Vitekta®),Genvoya® (contains elvitegravir, cobicistat, tenofovir alafenamidefumarte and emtricitabine), Stribild® (contains elvitegravir,cobicistat, tenofovir disoproxil fumarte and emtricitabine) raltegravir(Isentress®), Complera® (contains emtricitabine, tenofovir disoproxilfumarte, rilpivirine) and Atripla® (contains fixed-dose triplecombination of tenofovir, emtricitabine and efavirenz).

Between 5 and 10% of people with HIV are also infected with hepatitis Bvirus (often called co-infection). People with HIV are less likely tonaturally clear hepatitis B without treatment. People with HIV andhepatitis co-infection can have faster liver disease progression and maynot respond as well to hepatitis B treatment. However, having hepatitisB does not seem to make HIV disease worse. Hepatitis B virus (HBV)infection is the most common chronic viral infection in the world. Anestimated 2 billion people have been infected, and more than 350 millionare chronic carriers of the virus. HBV is transmitted through contactwith infected blood or semen.

Further, AIDS (HIV) and hepatitis B viruses are remarkably similar intheir sharing of reverse transcription, in their ancestral origins andcommon genetic elements, and in their modes of transmission. Both arehypermutable and exist as quasi-species due primarily to errors inreverse transcription, though there is severe restriction in thereplicative competence of most hepatitis B mutants. They differ in thelack of an integrase in hepatitis B virus and in their pathogenesis inthe infected host. HIV survives mainly by antigenic variability, immuneevasion, and impairment of immune function though viral regulatorycontrol elements seeking to restrict fatal damage to the host. HepatitisB virus survives primarily by mutation of e antigen/core genes thatdirectly obviate cytotoxic T cell destruction of infected liver cells,or indirectly limit destruction of infected cells through induction ofenergy in the cytotoxic T cell response.

Further, antiretroviral drugs such as lamivudine, adefovir, entecavirand tenofovir have been approved for the treatment of chronic HBVinfection.

Pharmacokinetic boosters or enhancers are used to boost theeffectiveness of antiretroviral drugs. When a pharmacokinetic booster orenhancer is co-administered with an antiretroviral drug, thepharmacokinetic enhancer interferes with the breakdown of theantiretroviral drug, which causes the antiretroviral drug to remain inthe body for a longer time and at a higher concentration.Pharmacokinetic boosters or enhancers specifically cause inhibition ofthe cytochrome P450 3A4 enzyme system leading to an increase in theplasma concentrations of the co-administered antiretroviral drugs.Protease Inhibitors are one such class of antiretroviral drugs thatgenerally exhibit high genetic barrier for drug resistance and hence dorequire a pharmacokinetic booster or enhancer to be co-administered. Outof all the approved drugs for the treatment of HIV, Ritonavir andCobicistat are termed as pharmacokinetic “boosters” or “enhancers”.Ritonavir is used because of its capacity to inhibit the drugmetabolizing enzyme cytochrome P450 (CYP) 3A4. Given in a low dose,ritonavir reduces the metabolism of protease inhibitors such aslopinavir and atazanavir, which are extensively metabolized by CYP3A4,thus enhancing the drug exposure. Cobicistat is also a strong inhibitorof CYP3A isozymes and increases plasma concentrations of drugs which aremetabolized by CYP3A such as protease inhibitors viz, atazanavir anddarunavir.

Besides ritonavir and cobicistat, there are many naturally occurringsubstances which are reported in literature and may be explored toimprove the pharmacokinetic activity of certain drugs.

These naturally occurring substances which act as bioenhancers arechemical entities that promote and augment the bioavailability of thedrugs which are mixed with them and do not exhibit synergistic effectwith the drug. Examples of these bioenhancers include piperine, garlic,Carum carvi, Cuminum cyminum, lysergol, naringin, quercetin, niaziridin,glycyrrhizin, stevia, cow urine, distillate ginger, etc.

These pharmacokinetic “boosters” or “enhancers” might reduce the cost ofantiviral therapy, reduce pill burden for patients, and/or reduce therisk of sub therapeutic antiviral concentrations (e.g., development ofresistance as well as enhance adherence to antiviral therapy).

However, this pharmacokinetic enhancement can be associated with its ownrisks. The precipitant drug, e.g., the booster or enhancer may have tobe administered in a dose that inhibits the elimination of the objectdrug as well as does not produce its own side effects.

Accordingly, the enhancer which usually is a potent inhibitor mayunintentionally inhibit the elimination of other drugs, leading tounwanted adverse effects. Also, if the dose of the enhancer is notcarefully adjusted, or is inadequate or in excess, it may ultimatelycause either a decrease or an increase of the object drug concentration.Hence considering these aspects, selection of the appropriate dose ofthe enhancer plays a vital role.

For example, although ritonavir has antiviral activity, it causesundesirable side effects, including gastrointestinal problems especiallychronic diarrhea and lipid abnormalities. Cobicistat was then developedto produce approximately the same degree of effect as ritonavir, butwithout antiviral activity or any other problematic side effects.

Cobicistat is a substrate for CYP3A4 (CYP2D6 is a minor pathway ofmetabolism) and inhibits its own metabolism. Further, cobicistat alsoinhibits P-glycoprotein (P-gp) and CYP2D6 and hence there are a numberof potential interactions that may occur with cobicistat.

Further, patients that are being treated for HIV are always at risk forinteractions with other non-HIV medication and cobicistat is known toexhibit key drug interactions with antacids, benzodiazepams,beta-blockers, calcium channel blockers, erectile dysfunction drugs,inhaled/injectable corticosteroids, statins, oral contraceptiveprogestins, rifampin and maraviroc.

The pharmacokinetic enhancers or boosters that are currently in useunintentionally inhibit the elimination of other drugs, leading tounwanted adverse effects. Also, the use of piperine and/or itsstructural analogs such as tetrahydropiperine, cis,trans-piperine,trans,cis-piperine, cis,cis-piperine and trans,trans-piperine are notknown to enhance the bioavailability of such anti-retroviral drugs.

Therefore, there remains a need to provide a combination therapy of apharmacokinetic booster or enhancer with such anti-retroviral drugs forthe treatment of HIV which reduces the dose of such anti-retroviraldrugs, side effects exhibited by these drugs as well as maintains theoptimal concentration of the same. Further, use of a naturally occurringpharmacokinetic booster or enhancer would eliminate or reduceinteractions with other non-HIV medications that would be concurrentlyadministered.

SUMMARY

In some embodiments, an object of the present invention is to provide acomposition comprising at least one anti-retroviral drug and at leastone pharmacokinetic booster or enhancer.

In some embodiments, another object of the present invention is toprovide a composition comprising at least one anti-retroviral drug andat least one pharmacokinetic booster or enhancer with reduced sideeffects.

In some embodiments, yet another object of the present invention is toprovide a composition comprising at least one anti-retroviral drug andat least one pharmacokinetic booster or enhancer with reduced druginteractions.

In some embodiments, another object of the present invention is toprovide a composition comprising at least one anti-retroviral drug andat least one pharmacokinetic booster or enhancer for once or twice a dayadministration.

In some embodiments, another object of the present invention is toprovide a composition comprising at least one anti-retroviral drug andat least one pharmacokinetic booster or enhancer with a reduced dose.

In some embodiments, yet another object of the present invention is toprovide a composition comprising at least one anti-retroviral drug andat least one pharmacokinetic booster or enhancer in the form of a kit.

In some embodiments, yet another object of the present invention is toprovide a method of prevention, treatment or prophylaxis of diseasescaused by viruses specifically caused by retroviruses, specificallyAcquired Immune Deficiency Syndrome or an HIV infection, the methodcomprising administering at least one anti-retroviral drug and at leastone pharmacokinetic booster or enhancer.

In some embodiments, yet another object of the present invention is toprovide a method of treatment of diseases caused by viruses specificallycaused by hepatitis B virus, the method comprising administering atleast one anti-retroviral drug and at least one pharmacokinetic boosteror enhancer.

In some embodiments, yet another object of the present invention is toprovide use of a pharmaceutical composition comprising at least oneanti-retroviral drug and at least one pharmacokinetic booster orenhancer for the treatment or prophylaxis of diseases caused by virusesspecifically caused by retroviruses, specifically Acquired ImmuneDeficiency Syndrome or an HIV infection.

In some embodiments, yet another object of the present invention is toprovide the use of a pharmaceutical composition comprising at least oneanti-retroviral drug and at least one pharmacokinetic booster orenhancer for the treatment of diseases caused by viruses specificallyhepatitis B virus.

According to an aspect of the present invention, provided is apharmaceutical composition comprising at least one anti-retroviral drugand at least one pharmacokinetic booster or enhancer and one or morepharmaceutically acceptable excipient.

According to another aspect of the invention, provided is a process forpreparing a pharmaceutical composition comprising at least oneanti-retroviral drug and at least one pharmacokinetic booster orenhancer with at least one or more pharmaceutically acceptableexcipients.

According to another aspect of the present invention, provided is amethod of treating diseases caused by viruses specifically caused byretroviruses, especially AIDS or an HIV infection, the method comprisingadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising at least one anti-retroviral drug and at leastone pharmacokinetic booster or enhancer according to the presentinvention to a patient in need thereof.

According to another aspect of the present invention there is provided amethod of treating diseases caused by viruses specifically caused byhepatitis B virus, the method comprising administering a therapeuticallyeffective amount of a pharmaceutical composition comprising at least oneanti-retroviral drug and at least one pharmacokinetic booster orenhancer according to the present invention to a patient in needthereof.

According to another aspect of the present invention provided is the useof a pharmaceutical composition comprising at least one anti-retroviraldrug and at least one pharmacokinetic booster or enhancer according tothe present invention in the manufacture of a medicament for thetreatment of diseases caused by viruses, specifically caused byretroviruses, especially AIDS or an HIV infection.

According to another aspect of the present invention there is providedthe use of a pharmaceutical composition comprising at least oneanti-retroviral drug and at least one pharmacokinetic booster orenhancer according to the present invention in the manufacture of amedicament for the treatment of diseases caused by viruses, specificallycaused by hepatitis B virus.

In some embodiments, an oral or injectable pharmaceutical composition isprovided comprising a therapeutically effective amount of at least oneanti-retroviral drug and a therapeutically effective amount of at leastone pharmacokinetic booster or enhancer or derivative thereof.

In some embodiments, an oral or injectable pharmaceutical composition isprovided comprising a therapeutically effective amount of at least oneanti-retroviral drug; a therapeutically effective amount of at least onepharmacokinetic booster or enhancer or derivative thereof; and one ormore pharmaceutically acceptable excipients comprising carriers,diluents, fillers, binders, lubricants, glidants, disintegrants, bulkingagents, flavorants or any combination thereof.

In some embodiments, a method of treating diseases caused byretroviruses or hepatitis B viruses in a patient in need of suchtreatment is provided, the method comprising: administering apharmaceutical composition comprising (i) a therapeutically effectiveamount of at least one anti-retroviral drug or an antiviral drug; (ii) atherapeutically effective amount of at least one pharmacokinetic boosteror enhancer or derivative thereof; and (iii) one or morepharmaceutically acceptable excipients comprising carriers, diluents,fillers, binders, lubricants, glidants, disintegrants, bulking agents,flavourants or any combination thereof.

In some embodiments, a method of making a pharmaceutical compositionthat enhances the bioavailability of an anti-retroviral drug isprovided, the method comprising: mixing a therapeutically effectiveamount of at least one anti-retroviral drug and a therapeuticallyeffective amount of at least one pharmacokinetic booster or enhancer orderivative thereof with one or more pharmaceutically acceptableexcipients to make the pharmaceutical composition.

In some embodiments, a kit for treating disease caused by retrovirusesor hepatitis B viruses is provided, the kit comprising a therapeuticallyeffective amount of at least one anti-retroviral drug and atherapeutically effective amount of at least one pharmacokinetic boosteror enhancer or derivative thereof, wherein the at least oneanti-retroviral drug is in a separate composition from the at least onepharmacokinetic booster or enhancer or derivative thereof.

In some embodiments, a method of enhancing the bioavailability of anoral anti-retroviral drug is provided, the method comprising: providinga therapeutically effective amount of at least one anti-retroviral drugand providing a therapeutically effective amount of at least onepharmacokinetic booster or enhancer or derivative thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a bar graph of results from a unidirectional assayshowing permeability of tenofovir alafenamide fumarate (TAF) andtenofovir disoproxil fumarate (TDF). TDF and TAF were observed to be lowto moderately permeable drugs.

FIG. 2 depicts a bar graph of results from a bidirectional assay ofDigoxine 10 μM, Digoxine 10 μM (A-B)+Piperine 10 μM, TAF 100 μM(VRD-1063/16/187), TAF 100 μM (VRD-1063/16/187)+Piperine 0.1 μM and TAF100 μM (VRD-1063/16/187)+Piperine 10 μM. Results showed that TAFabsorption is increased with piperine by decreasing the efflux ratio ofTAF.

FIG. 3 depicts a bar graph of results from a bidirectional assay ofDigoxin 10 μM, Dolutegravir 5 μM, Dolutegravir 5 μM+Piperine 1 μM,Dolutegravir 5 μM+Piperine 10 μM, Dolutegravir 5 μM+Verapamil 1 μM, andDolutegravir 5 μM+Verapamil 10 μM.

FIG. 4 depicts a bar graph of results from a bidirectional assay ofDigoxin 10 μM, Darunavir 40 μM, Darunavir 40 μM+Piperine 1 μM, Darunavir40 μM+Piperine 10 μM, Darunavir 40 μM+Cobicistat 10 μM, and Darunavir 40μM+Cobicistat 100 μM. Results showed that absorption of Darunavir isincreased with piperine by decreasing the efflux ratio of TAF.

FIG. 5 depicts a bar graph of results from a bidirectional assay ofDigoxin 10 μM TDF 200 μM, TDF 100 μM, TDF 100 μM+Piperine 10 μM, and TDF100 μM+Tetrahydro Piperine 10 μM. Results showed that absorption of TDFis increased with piperine by decreasing the efflux ratio, absorption ofTDF is increased with tetrahydropiperine by decreasing the efflux ratio,and comparable improvement in permeability of TDF was seen by bothPiperine and tetrahydropiperine.

FIG. 6 depicts a bar graph of plasma concentrations of tenofovir for TDF300 mg and TDF 300 mg+Piperine 20 mg at different time points.

FIG. 7 depicts a bar graph of plasma concentrations of tenofovir for TDF300 mg and TDF 300 mg+Piperine 20 mg at different time points.

FIG. 8 depicts time dependent plasma concentrations of tenofovir for 300mg TDF, 300 mg TDF+20 mg piperine and 150 mg TDF+20 mg piperine.

It is to be understood that the figures are not drawn or photographed toscale. Further, the relation between objects in a figure may not be toscale, and may in fact have a reverse relationship as to size. Thefigures are intended to bring understanding and clarity to the structureof each object shown, and thus, some features may be exaggerated inorder to illustrate a specific feature of a structure.

DETAILED DESCRIPTION

For the treatment of diseases caused by retroviruses or hepatitis Bvirus, especially AIDS, an HIV infection or hepatitis B, it is essentialthat the maximum amount of the drug reaches the site of action. Mostantiretroviral drugs either have poor solubility and/or poorpermeability which deteriorates the bioavailability of the drug to amajor extent.

The inventors of the present invention have found ways to address thebioavailability problems of such anti-retroviral drugs. In particular,the inventors have found that, the bioavailability properties of thesedrugs can be improved by using a pharmacokinetic booster or enhancer.

Enhanced bioavailability of an anti-viral drug is disclosed in severalreferences. Role of Piperine As A Bioavailability Enhancer, UMESH KPATIL et al International Journal of Recent Advances in PharmaceuticalResearch October 2011; 4: 16-23 discloses piperine as a bioavailabilityenhancer.

WO2004067018 discloses the use of extracts of Carum carvi asbioenhancers, either alone or in combination with piperine or Zinzeberoficinale extract to improve the bioavailability of zidovudine.

Natural Bioenhancers: An overview, Deepthi V. Tatiraju et al, Journal ofPharmacognosy and Phytochemistry 2013; 2 (3): 55-60. This articlediscloses the combination of piperine with nevirapine, wherein piperineenhanced the bioavailability of nevirapine.

Oral bioavailability enhancement of an anti-viral drug using an herbalbio-enhancer, Mohammad Asif, a dissertation submitted to the GanpatUniversity. This article discloses the combination of Piperine withefavirenz, wherein piperine enhanced the bioavailability of efavirenz.

Bioenhancement effect of piperine and ginger oleo resin on thebioavailability of atazanavir, Swati Prakash et al, InternationalJournal of Pharmacy and Pharmaceutical Sciences Vol 7, Issue 10, 2015.This article discloses the combination of piperine with atazanvir,wherein piperine enhanced the bioavailability of atazanvir.

WO03084462 discloses the process for manufacturing pharmaceuticalcomposition containing antiretroviral protease inhibitor such asindinavir, saquinavir, amprenavir, nelfinavir, lopinavir and piperine ina single pharmaceutical composition.

In some embodiments, the anti-retroviral drugs, according to the presentinvention, include but are not limited to Nucleoside ReverseTranscriptase Inhibitors (NRTI), Non-Nucleoside Reverse TranscriptaseInhibitors (NNRTI), Nucleotide Analog Reverse-Transcriptase Inhibitors,Protease Inhibitors (PI), Integrase Inhibitors, Fusion Inhibitors, CCR5Inhibitors, Monoclonal Antibodies, Glycoprotein Inhibitors and anycombinations thereof.

In one embodiment, the Nucleoside Reverse Transcriptase Inhibitors(NRTI) and Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTI)include but are not limited to lamivudine, abacavir, zidovudine,emtricitabine didanosine, stavudine, lobucavir, entecavir, apricitabinecensavudine, zalcitabine, dexelvucitabine, alovudine, efavirenz,amdoxovir, elvucitabine, festinavir, racivir, lersivirine, rilpivirine,etravirine, stampidine, Doravirine, Dapivirine.

In some embodiments, preferably, the Nucleoside Reverse TranscriptaseInhibitors (NRTI) and Non-Nucleoside Reverse Transcriptase Inhibitors(NNRTI) are abacavir, didanosine. Preferably the dose of abacavir rangesfrom about 3 mg to about 300 mg, and didanosine ranges from about 2 mgto about 200 mg for twice a day administration.

In another embodiment, protease inhibitors include but are not limitedto lopinavir, ritonavir, saquinavir, nelfinavir, amprenavir, indinavir,nelfinavir, atazanavir, lasinavir, palinavir, tirpranavir,fosamprenavir, darunavir, or tipranavir. Preferably, the proteaseinhibitors are tirpranavir, darunavir. Preferably the dose of tipranavirranges from about 5 mg to about 500 mg, and darunavir ranges from about1 mg to about 800 mg for twice a day administration. In someembodiments, the darunavir dose ranges from about 1 mg to about 500 mg,from about 20 mg to about 500 mg, from about 25 mg to about 500 mg, fromabout 30 mg to about 500 mg, from about 35 mg to about 500 mg, fromabout 25 mg to about 35 mg, from about 50 mg to about 400 mg, or fromabout 100 mg to about 300 mg for twice a day administration. In someembodiments, the darunavir dose ranges from about 1 mg, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570,580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710,720, 730, 740, 750, 760, 770, 780, 790 to about 800 mg for once a day ortwice a day administration. Each dose can be in one or more unit dosageforms, as described herein.

In another embodiment, integrase inhibitors include but are not limitedto dolutegravir, elvitegravir, raltegravir, bictegravir, cabotegravir.Preferably, the integrase inhibitors are elvitegravir, dolutegravir,raltegravir. Preferably the dose of Dolutegravir ranges from about 1 mgto about 50 mg, Elvitegravir ranges from about 1 mg to about 150 mg foronce a day administration and that of Raltegravir ranges from about 4 mgto about 400 mg for once a day administration. In some embodiments, thedolutegravir dose ranges from about 5 mg to about 50 mg, from about 20mg to about 50 mg, from about 25 mg to about 50 mg, from about 25 mg toabout 45 mg, from about 30 mg to about 50 mg, from about 30 mg to about40 mg, or from about 35 mg to about 50 mg for twice a dayadministration. In some embodiments, the dolutegravir dose ranges fromabout 1 mg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 to about 50 mg foronce a day or twice a day administration. Each dose can be in one ormore unit dosage forms, as described herein.

In another embodiment, Fusion inhibitors include but are not limited toMaraviroc, Enfuvirtide, Griffithsin, Aplaviroc, Vicriviroc, Plerixafor,Fostemsavir, Albuvirtide.

In another embodiment, CCR5 inhibitors include but are not limited toAplaviroc, Vicriviroc, Maraviroc, Cenicriviroc.

In another embodiment, Monoclonal Antibodies include but are not limitedto Ibalizumab.

In another embodiment, Glycoprotein Inhibitors include but are notlimited to Sifuvirtide.

In another embodiment, Nucleotide Analog Reverse-TranscriptaseInhibitors include but are not limited to tenofovir alafenamidefumarate, tenofovir disoproxil fumarate and adefovir. Preferably, theNucleotide Analog Reverse-Transcriptase Inhibitors are tenofoviralafenamide fumarate and tenofovir disoproxil fumarate. In someembodiments, the tenofovir alafenamide fumarate dose ranges from about 1mg to about 25 mg, from about 2.5 mg to about 25 mg, from about 5 mg toabout 20 mg, or from about 5 mg to about 15 mg for twice a dayadministration. In some embodiments, the tenofovir alafenamide fumaratedose ranges from about 1 mg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 to about 25 mg for once a day ortwice a day administration. In some embodiments, the tenofovirdisoproxil fumarate dose ranges from about 1 mg to about 300 mg, fromabout 1 mg to about 150 mg, from about 75 mg to about 250 mg, from about100 mg to about 200 mg, or from about 120 to about 180 mg for twice aday administration. In some embodiments, the tenofovir disoproxilfumarate dose ranges from about 1 mg, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 260, 270, 280, 290 to about 300 mg foronce a day or twice a day administration. Each dose can be in one ormore unit dosage forms, as described herein.

The term “Anti-retroviral drug” and “Pharmacokinetic booster orenhancer” is used in broad sense to include not only “Anti-retroviraldrug” per se and “Pharmacokinetic booster or enhancer” per se but alsoits pharmaceutically acceptable derivatives thereof. Suitablepharmaceutically acceptable derivatives include pharmaceuticallyacceptable salts, pharmaceutically acceptable solvates, pharmaceuticallyacceptable hydrates, pharmaceutically acceptable anhydrates,pharmaceutically acceptable enantiomers, pharmaceutically acceptableesters, pharmaceutically acceptable isomers, pharmaceutically acceptablepolymorphs, pharmaceutically acceptable prodrugs, pharmaceuticallyacceptable tautomers, pharmaceutically acceptable complexes etc.

The term “pharmacokinetic booster or enhancer” is an alkaloid. In someembodiments, the pharmacokinetic booster or enhancer comprises piperine,tetrahydropiperine, cis-piperine, trans-piperine, cis-trans piperine,trans,cis-piperine, cis,cis-piperine, trans,trans-piperine or acombination thereof. More preferably, the pharmacokinetic booster orenhancer is piperine or tetrahydropiperine and its analogs orderivatives. In some embodiments, the pharmacokinetic booster orenhancer increases plasma concentrations of the anti-retroviral drug by10%, 20, 30, 40, 50, 60, 70, 80, 90, 100% or higher in comparison towhen the pharmacokinetic booster or enhancer is not used.

The term “injectable” is a mode of administering the pharmaceuticalcomposition. The pharmaceutical composition can be administered in avariety of ways. In humans, the pharmaceutical composition can beadministered by the parenteral route. For example, the pharmaceuticalcomposition can be administered intravenously (e.g., intravenousinjection), subcutaneously, intradermally, or by intramuscularinjection. Intravenous administration can be accomplished by mixing thepharmaceutical composition in a suitable pharmaceutical carrier(vehicle) or excipient as understood by practitioners in the art.Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the pharmaceuticalcomposition, which can be formulated to be isotonic with the blood ofthe patient.

The term “therapeutically effective amount” or “effective amount” issuch that when administered, the pharmaceutical composition results inthe inhibition of a virus or disease. The dosage administered to apatient can be as single or multiple doses depending upon a variety offactors, including the drug's administered pharmacokinetic properties,the route of administration, patient conditions and characteristics(sex, age, body weight, health, size, etc.), and extent of symptoms,concurrent treatments, frequency of treatment and the effect desired.

The term “treatment” or “treating” of a disease, virus or conditionrefers to executing a protocol that may include administering one ormore drugs to a patient, in an effort to alleviate signs or symptoms ofthe disease, virus or condition. Alleviation can occur prior to signs orsymptoms of the disease, virus or condition appearing, as well as aftertheir appearance. Thus, treating or treatment includes reducing,preventing or prevention of the disease, virus or condition. Inaddition, treating or treatment does not require complete alleviation ofsigns or symptoms, does not require a cure, and specifically includesprotocols that have only a marginal effect on the patient.

The fruit of black pepper (Piper nigrum L.) and long pepper (Piperlongum L.) are both important medicinal herbs in Ayurvedic and Unani(traditional Indian) systems of medicine, wherein the remedy generallyconsists of mixtures of herbs. A wide range of the medicinal uses ofblack pepper are known and have been documented including its use in thetreatment of leucoderma.

Piperine, can be the pharmacokinetic booster or enhancer. Piperine, themajor alkaloid found in the fruit of black pepper (Piper nigrum L;Piperaceae), stimulates the replication of melanocytes and induces theformation of melanocytic dendrites. Piperine is expected to cause therepopulation of vitiligo patches through a stimulatory effect onperilesional and follicular melanocytes.

Piperine is chemically known as (1-2E, 4E-piperinoyl-piperidine) and isstructurally represented as shown below.

Piperine may enhance the drug bioavailability by promoting rapidabsorption of drugs and nutrients by increasing blood supply to thegastrointestinal tract, decreasing hydrochloric acid secretion toprevent the breakdown of some drugs, increasing the emulsifying contentof the gut, increasing enzymes like γ-glutamyl transpeptidase whichparticipate in active and passive transport of nutrients to theintestinal cells.

Piperine may increase the drug bioavailability by inhibiting enzymeswhich participate in the biotransformation of drugs and thus preventingtheir inactivation and elimination. It also inhibits p-glycoprotein, the‘pump’ protein that removes substances from cells and can decrease theintestinal production of glucuronic acid, thereby permitting moresubstances to enter the body in active form.

Piperine has also been reported to occur in other Piper species i.e. P.acutisleginum, album, argyrophylum, attenuatum, aurantiacum, betle,callosum, chaba, cubeba, guineense, hancei, khasiana, iongum,macropodum, nepalense, novae hollandiae, peepuloides, retrokacturn, andsylvaticum.

Tetrahydropiperine is a structural analog of Piperine. The two doublebonds at position 2 and 4 are saturated to give a tetrahydro analog.Tetrahydropiperine is chemically known as5-(1,3-benzodioxol-5-yl)-1-piperidin-1-ylpentan-1-one and isstructurally represented as shown below.

Tetrahydropiperine occurs like piperine naturally in black pepper (about0.7% in black pepper oleoresin). Tetrahydropiperine can be synthesizedfrom piperine which is previously extracted from black pepper oleoresin.

The term “analogs or derivatives” of tetrahydropiperine is used in broadsense to include alkyltetrahydropiperines, e.g. methyltetrahydropiperineor ethyltetrahydropiperine, dialkyltetrahydropiperines, e.g.dimethyltetrahydropiperine or diethyltetrahydropiperine, alkoxylatedtetrahydropiperine, e.g. methoxy tetrahydropiperine, hydroxylatedtetrahydropiperine, e.g.1-[(5,3-benzodioxyl-5-yl)-1-hydroxy-2,4-pentadienyl]-piperine,1-[(5,3-benzodioxyl-5-yl)-1-methoxy-2,4-pentadienyl]-piperine,halogenated tetrahydropiperine, e.g.1-[(5,3-benzodioxyl-5-yl)-1-oxo-4-halo-2-pentenyl]-piperine and1-[(5,3-benzodioxyl-5-yl)-1-oxo-2-halo-4-pentenyl]-piperine,dihydropiperine, alkyldihydropiperines, e.g. methyldihydropiperine orethyldihydropiperine, dialkyldihydropiperines, e.g.dimethyldihydropiperine or diethyldihydropiperine, alkoxylateddihydropiperine, e.g. methoxy dihydropiperine, and halogenateddihydropiperine and their pharmaceutically acceptable salts,pharmaceutically acceptable solvates, pharmaceutically acceptablehydrates, pharmaceutically acceptable anhydrates, pharmaceuticallyacceptable enantiomers, pharmaceutically acceptable esters,pharmaceutically acceptable isomers, pharmaceutically acceptablepolymorphs, pharmaceutically acceptable prodrugs, pharmaceuticallyacceptable tautomers, pharmaceutically acceptable complexes etc.

In some embodiments, preferably the dose of piperine ranges from about0.5 mg to about 400 mg and the dose of tetrahydropiperine ranges fromabout 0.5 mg to about 400 mg. In some embodiments, the dose of thepiperine and/or the tetrahydropiperine ranges from about 0.5 mg, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, to about 400 mg. In some embodiments, the ratio of the atleast one anti-retroviral drug to the at least one pharmacokineticbooster or enhancer is from about 100:1 to about 1:1 by weight.

Preferably, the pharmaceutical composition may be provided in dosageforms such as but not limited to, unit dosage forms including tablets,capsules (filled with powders, pellets, beads, mini-tablets, pills,micro-pellets, small tablet units, multiple unit pellet systems (MUPS),disintegrating tablets, dispersible tablets, granules, and microspheres,multiparticulates), sachets (filled with powders, pellets, beads,mini-tablets, pills, micro-pellets, small tablet units, MUPS,disintegrating tablets, dispersible tablets, granules, and microspheres,multiparticulates), powders for reconstitution, transdermal patches andsprinkles, however, other dosage forms such as controlled releaseformulations, lyophilized formulations, lyophilized powder, modifiedrelease formulations, delayed release formulations, extended releaseformulations, pulsatile release formulations, dual release formulationsand the like. Liquid, liquid injectable or semisolid dosage form(liquids, suspensions, solutions, dispersions, ointments, creams,emulsions, microemulsions, sprays, patches, spot-on), injectionpreparations, parenteral, topical, inhalations, buccal, nasal etc. mayalso be envisaged under the ambit of the invention. In some embodiments,the pharmaceutical composition is administered via a syrup. A syrup maybe made by adding the active compound to a concentrated aqueous solutionof a sugar, for example sucrose, to which may also be added anyaccessory ingredient(s). Such accessory ingredients may includeflavorings, suitable preservatives, an agent to retard crystallizationof the sugar, and an agent to increase the solubility of any otheringredient, such as polyhydric alcohol, for example, glycerol orsorbitol.

In some embodiments, a unit dosage from, such as a tablet may be made bycompression or molding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine, with the active compound being in a free-flowing formsuch as a powder or granules which is optionally mixed with a binder,disintegrant, lubricant, inert diluent, surface active agent ordispersing agent. Molded tablets comprised with a suitable carrier maybe made by molding in a suitable machine.

The pharmaceutical compositions of the present invention comprise atleast one anti-retroviral drug and piperine or tetrahydropiperine. Theseactive ingredients are formulated for simultaneous, separate orsequential administration. When the active ingredients are administeredsequentially, either at least one anti-retroviral drug orpiperine/tetrahydropiperine, may be administered first. Whenadministration is simultaneous, the active ingredients may beadministered either in the same or different pharmaceuticalcompositions. Adjunctive therapy, e.g., where one active ingredient isused as the primary treatment and the other active ingredient(s) is/areused to assist that primary treatment is also an embodiment of thepresent invention.

Accordingly, there is provided a product comprising at least oneanti-retroviral drug and piperine or tetrahydropiperine as a combinedpreparation for simultaneous, separate or sequential use for treatmentof diseases caused by retroviruses or hepatitis B virus, especially AIDSor an HIV infection, or hepatitis B.

In some embodiments, the pharmaceutical compositions of the presentinvention comprise tenofovir disproxil fumarate and piperine for thetreatment of diseases caused by retroviruses, especially Acquired ImmuneDeficiency Syndrome or an HIV infection.

According to a preferred embodiment, the pharmaceutical compositions ofthe present invention comprise tenofovir disproxil fumarate and piperinein a ratio from about 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 8:1, 6:1,5:1, 4:1, 3:1, 2:1, to about 1:1 by weight.

In some embodiments, the pharmaceutical compositions of the presentinvention comprise tenofovir alafenamide fumarate and piperine for thetreatment of diseases caused by retroviruses, especially AIDS or an HIVinfection.

According to a preferred embodiment, the pharmaceutical compositions ofthe present invention comprise tenofovir alafenamide fumarate andpiperine in a ratio from about 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 8:1,6:1, 5:1, 4:1, 3:1, 2:1, to about 1:1 by weight.

In some embodiments, the pharmaceutical compositions of the presentinvention comprise dolutegravir and piperine for the treatment ofdiseases caused by retroviruses, especially Acquired Immune DeficiencySyndrome or an HIV infection.

According to a preferred embodiment, the pharmaceutical compositions ofthe present invention comprise dolutegravir and piperine in a ratio fromabout from about 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 8:1, 6:1, 5:1,4:1, 3:1, 2:1, to about 1:1 by weight.

In some embodiments, the pharmaceutical compositions of the presentinvention comprise darunavir and piperine for the treatment of diseasescaused by retroviruses, especially Acquired Immune Deficiency Syndromeor an HIV infection.

According to a preferred embodiment, the pharmaceutical compositions ofthe present invention comprise darunavir and piperine in a ratio fromabout 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1,to about 1:1 by weight.

In some embodiments, the pharmaceutical compositions of the presentinvention comprise tenofovir disproxil fumarate and piperine fortreatment of diseases caused by hepatitis B virus.

According to a preferred embodiment, the pharmaceutical compositions ofthe present invention comprise tenofovir disproxil fumarate and piperinein a ratio from about 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 8:1, 6:1,5:1, 4:1, 3:1, 2:1, to about 1:1 by weight.

In some embodiments, the pharmaceutical compositions of the presentinvention comprise tenofovir alafenamide fumarate and piperine fortreatment of diseases caused by hepatitis B virus.

According to a preferred embodiment, the pharmaceutical compositions ofthe present invention comprise tenofovir alafenamide fumarate andpiperine in a ratio from about 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 8:1,6:1, 5:1, 4:1, 3:1, 2:1, to about 1:1 by weight.

In some embodiments, the pharmaceutical compositions of the presentinvention comprise tenofovir disproxil fumarate and tetrahydropiperinefor the treatment of diseases caused by retroviruses, especiallyAcquired Immune Deficiency Syndrome or an HIV infection.

According to a preferred embodiment, the pharmaceutical compositions ofthe present invention comprise tenofovir disproxil fumarate andtetrahydropiperine in a ratio from about 100:1, 50:1, 40:1, 30:1, 20:1,10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, to about 1:1 by weight.

In some embodiments, the pharmaceutical compositions of the presentinvention comprise tenofovir alafenamide fumarate and tetrahydropiperinefor the treatment of diseases caused by retroviruses, especiallyAcquired Immune Deficiency Syndrome or an HIV infection.

According to a preferred embodiment, the pharmaceutical compositions ofthe present invention comprises tenofovir alafenamide fumarate andtetrahydropiperine in a ratio from about 100:1, 50:1, 40:1, 30:1, 20:1,10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, to about 1:1 by weight.

In some embodiments, the pharmaceutical compositions of the presentinvention comprise dolutegravir and tetrahydropiperine for the treatmentof diseases caused by retroviruses, especially Acquired ImmuneDeficiency Syndrome or an HIV infection.

According to a preferred embodiment, the pharmaceutical compositions ofthe present invention comprises dolutegravir and tetrahydropiperine in aratio from about 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 8:1, 6:1, 5:1,4:1, 3:1, 2:1, to about 1:1 by weight.

In some embodiments, the pharmaceutical compositions of the presentinvention comprise darunavir and tetrahydropiperine for the treatment ofdiseases caused by retroviruses, especially Acquired Immune DeficiencySyndrome or an HIV infection.

According to a preferred embodiment, the pharmaceutical compositions ofthe present invention comprises darunavir and tetrahydropiperine in aratio from about 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 8:1, 6:1, 5:1,4:1, 3:1, 2:1, to about 1:1 by weight.

In some embodiments, the pharmaceutical compositions of the presentinvention comprise tenofovir disproxil fumarate and tetrahydropiperinefor treatment of diseases caused by hepatitis B virus.

According to a preferred embodiment, the pharmaceutical compositions ofthe present invention comprises tenofovir disproxil fumarate andtetrahydropiperine in a ratio from about 100:1, 50:1, 40:1, 30:1, 20:1,10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, to about 1:1 by weight.

In some embodiments, the pharmaceutical compositions of the presentinvention comprise tenofovir alafenamide fumarate and tetrahydropiperinefor treatment of diseases caused by hepatitis B virus.

According to a preferred embodiment, the pharmaceutical compositions ofthe present invention comprises tenofovir alafenamide fumarate andtetrahydropiperine in a ratio from about 100:1, 50:1, 40:1, 30:1, 20:1,10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, to about 1:1 by weight.

In some embodiments, when the pharmacokinetic booster or enhancer orderivative thereof is administered with the anti-retroviral drug in thepharmaceutical composition, a dosing frequency of the at least oneanti-retroviral drug that is administered to a patient is reduced. Insome embodiments, the at least one pharmacokinetic booster or enhanceror derivative thereof increases the bioavailability of the at least oneanti-retroviral drug from about 10% to about 100%, from about 10% toabout 70%, from about 10% to about 50%, from about 10% to about 30%, orfrom about 10% to about 20%. In some embodiments, the at least onepharmacokinetic booster or enhancer or derivative thereof increases thebioavailability of the at least one anti-retroviral drug from about 10%,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

The inventors of the present invention have also found that thebioavailability properties of anti-retroviral drugs may also be improvedby nanosizing. In some embodiments, the pharmaceutical composition isadministered via nanoparticles having a size of about 1 nanometer (nm)to about 50 nm. In some embodiments, the nanoparticles have a size offrom about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nm.

In some embodiments, suitable excipients may be used for formulating thedosage forms according to the present invention such as, but not limitedto, surface stabilizers or surfactants, viscosity modifying agents,polymers including extended release polymers, stabilizers, disintegrantsor super disintegrants, diluents, plasticizers, binders, glidants,lubricants, sweeteners, flavoring agents, anti-caking agents,opacifiers, anti-microbial agents, antifoaming agents, emulsifiers,buffering agents, coloring agents, carriers, fillers, anti-adherents,solvents, taste-masking agents, preservatives, antioxidants, textureenhancers, channeling agents, coating agents or combinations thereof.

In some embodiments, when the pharmaceutical composition is provided inunit dosage forms, as discussed above, the unit dosage form can beuncoated or coated.

These and other aspects of the present application will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the applicationbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1

TABLE 1 Ingredient Tab (%) Dry mix Tenofovir Alafenamide Fumarate0.1%-10%  Emtricitabine 25%-40%  Piperine 0.1%-10%  Lactose 5%-40%Colloidal silicon dioxide 1%-10% Microcrystalline Cellulose 1%-15%Croscarmellose Sodium 1%-10% Magnesium Stearate 0.5%-10%  Blending &Lubrication Colloidal silicon dioxide 1%-10% Microcrystalline Cellulose1%-15% Croscarmellose Sodium 1%-10% Magnesium Stearate 0.5%-10%  FilmCoating Opadry 1%-10%

Process:

1) Tenofovir alafenamide fumarate, emtricitabine, piperine, lactose,colloidal silicon dioxide, microcrystalline cellulose and croscarmellosesodium were dry mixed in a suitable blender.2) The blend obtained in step (1) was lubricated with magnesium stearateand was compacted and dry granulated.3) The granules obtained in step (3), colloidal silicon dioxide,microcrystalline cellulose and croscarmellose sodium were mixed to forma blend.4) The blend obtained in step (3) was compressed to form tablets andcoated with Opadry.

Example 2

TABLE 2 Ingredient Tab wt. % Dry mix Tenofovir Alafenamide Fumarate0.1%-10%  Piperine 0.1%-10%  Lactose 5%-40% Microcrystalline Cellulose1%-15% Croscarmellose Sodium 1%-10% Lubrication Magnesium Stearate0.5%-10%  Film Coating Opadry 1%-10%

Process:

1) Tenofovir alafenamide fumarate, piperine, lactose, colloidal silicondioxide, microcrystalline cellulose and croscarmellose sodium were drymixed in a suitable blender.2) The blend obtained in step (1) was lubricated with magnesiumstearate, compressed to form tablets and coated with Opadry.

Example 3

TABLE 3 Ingredient Tab wt. % Dry mix Tenofovir Alafenamide Fumarate0.1%-10%  Emtricitabine 25%-40%  Piperine 0.1%-10%  Elvitegravir10%-30%  Lactose 5%-40% Colloidal silicon dioxide 1%-10%Microcrystalline Cellulose 1%-15% Croscarmellose Sodium 1%-10% MagnesiumStearate 1%-10% Blending & Lubrication Colloidal silicon dioxide 1%-10%Microcrystalline Cellulose 1%-15% Croscarmellose Sodium 1%-10% MagnesiumStearate 1%-10% Film Coating Opadry 1%-10%

Process:

1) Tenofovir alafenamide fumarate, emtricitabine, piperine,elvitegravir, lactose, colloidal silicon dioxide, croscarmellose sodiumand microcrystalline cellulose were dry mixed to obtain a blend.2) The blend obtained in step (1) was lubricated with magnesiumstearate, compacted, sized compressed to form tablets.

Example 4

TABLE 4 Ingredient Tab wt. % Dry mix Tenofovir Disoproxil Fumarate10%-40%  Emtricitabine 25%-40%  Piperine 0.1%-10%  Elvitegravir 10%-30% Lactose 5%-40% Colloidal silicon dioxide 1%-10% MicrocrystallineCellulose 1%-15% Croscarmellose Sodium 1%-10% Magnesium Stearate 1%-10%Blending & Lubrication Colloidal silicon dioxide 1%-10% MicrocrystallineCellulose 1%-15% Croscarmellose Sodium 1%-10% Magnesium Stearate 1%-10%Film Coating Opadry 1%-10%

Process:

Tenofovir disoproxil fumarate, emtricitabine, piperine, elvitegravir,lactose, colloidal silicon dioxide, croscarmellose sodium andmicrocrystalline cellulose were dry mixed to obtain a blend.2) The blend obtained in step (1) was lubricated with magnesiumstearate, compacted, sized compressed to form tablets.

Example 5

TABLE 5 Sr. No. Ingredients Qty/Unit (mg) 1. Dolutegravir Sodium 5-50 2.Piperine 0.1-50  3. Mannitol 10-75  4. Povidone 2-20 5. Sodium StarchGlycolate 5-20 6. Mannitol 50-150 7. Microcrystalline cellulose 20-1008. Colloidal silicon dioxide 0.1-2   9. Sodium Stearyl Fumarate 0.5-10 10. Coating pre-mix 1-20

Process:

Dolutegravir sodium, Piperine, Mannitol were dry mixed and Povidone wasdissolved in water.2) The dry mix obtained in step (1) was granulated and the granulesobtained were milled.3) The granules obtained in step (2) were blended with mannitol, sodiumstarch glycolate, microcrystalline cellulose and colloidal silicondioxide.4) The blend obtained in step (3) was lubricated with Sodium stearylfumarate, compressed and coated.

Example 6

TABLE 6 Sr. No. Ingredients Mg/Tab 1 Darunavir Hydrate 50-651 2 Piperine 5-100 3 Microcrystalline Cellulose 50--540 4 Crospovidone 0-25 5Colloidal silicon dioxide 0.5-25   6 Magnesium Stearate 0.1-10   FilmCoating 7 Opadry 5-50 8 Purified Water qs

Process:

1) Darunavir hydrate, Piperine, microcrystalline cellulose, Crospovidone& Colloidal silicon dioxide were sifted and mixed.2) The dry mix obtained in step (1) was granulated and lubricated withmagnesium stearate.3) The granules obtained in step (2) were compressed and coated withOpadry.

Example 7

TABLE 7 Sr. No. Ingredients Qty/Unit (mg) 1 Darunavir Hydrate 50-870 2Piperine 0.5-250  3 Povidone 0.1-15  4 Microcrystalline Cellulose 10-2805 Crospovidone 0.5-35  6 Colloidal Silicon Dioxide 0.-7.0 7 MagnesiumStearate 0.1-9.0  Film Coating 8 Opadry 10-50  9 Purified Water q.s.

Process:

1) Darunavir hydrate, Piperine, povidone, microcrystalline cellulose,Crospovidone & Colloidal silicon dioxide were sifted and mixed.2) The dry mix obtained in step (1) was granulated and lubricated withmagnesium stearate.3) The granules obtained in step (2) were compressed and coated withOpadry.

Example 8

TABLE 8 Sr. No Ingredients Qty/Unit (mg) 1 Darunavir Ethanolate 50-651 2Piperine  5-300 2 Silicified Microcrystalline Cellulose 10-551 3Colloidal Silicon Dioxide 0-25 4 Crospovidone 0.2-15  5 MagnesiumStearate 0.1-10  Film Coating 7 Opadry 5-50 8 Purified Water q.s. Total

Process:

Darunavir ethnolate, Piperine, povidone, silicified microcrystallinecellulose, Crospovidone & Colloidal silicon dioxide were sifted andmixed.2) The dry mix obtained in step (1) was granulated and lubricated withmagnesium stearate.3) The granules obtained in step (2) were compressed and coated withOpadry.

Example 9

TABLE 9 Sr. No. Ingredients Qty/Unit (mg) 1. Darunavir Ethanolate 50-8702. Piperine  5-400 3. Hydroxy propyl methyl cellulose 0.1-15  4.Silicified Microcrystalline cellulose 10-180 5. Crospovidone 0.1-40  6.Colloidal silicon dioxide  0-5.0 7. Magnesium stearate 0.1-10  Filmcoating 8. Opadry 5-40 9. Purified water q.s.

Process:

1) Darunavir ethnolate, Piperine, Hydroxy propyl methyl cellulose,silicified microcrystalline cellulose, Crospovidone & Colloidal silicondioxide were sifted and mixed.2) The dry mix obtained in step (1) was granulated and lubricated withmagnesium stearate.3) The granules obtained in step (2) were compressed and coated withOpadry.

Example 10

TABLE 10 Sr. No. Ingredients Qty/Tab (mg) Dry Mix (Lamivudine Part) 1.Lamivudine 300.00 2. Microcrystalline cellulose 50.80 3. Croscarmellosesodium 22.50 4. Pregelatinized starch 18.00 5. Magnesium Stearate 2.50Dry Mix (Tenofovir Disoproxil Fumarate Part) 6. Tenofovir DisoproxilFumarate 100.00 7 Piperine 20.00 8. Microcrystalline cellulose 45.20 9.Croscarmellose sodium 22.50 10. Magnesium Stearate 2.50 Blending andLubrication 11. Croscarmellose sodium 30.00 12. Microcrystallinecellulose 100.00 13. Magnesium Stearate 6.00 Total 720.00 Seal Coating14. Hypromellose 5.00 15. Isopropyl Alcohol q.s 16. Purified water q.sTotal 725.00 Film Coating 17. Opadry II 85G18490 White 22.00 18.Purified water q.s Final Tablet weight 747.00

Process:

1) Lamivudine, Microcrystalline cellulose, Croscarmellose sodium,Pregelatinised starch and magnesium stearate was blended and compactedinto granular mass.2) Tenofovir disoproxil fumarate, Piperine, Microcrystalline cellulose,Croscarmellose sodium and magnesium stearate was blended and compactedinto granular mass.3) Microcrystalline cellulose, Croscarmellose sodium and magnesiumstearate was mixed with the blends obtained in step (1) and step (2) andcompressed to form tablets with seal coating followed by film coating.

Example 11

TABLE 11 Sr. No. Ingredients Qty/Tab (mg) Dry Mix (Emtricitabine, TDF &Piperine part) 1. Emtricitabine 200.000 2. Tenofovir Disoproxil Fumarate100.000 3. Piperine 20.00 4. Lactose monohydrate 80.000 5.Croscarmellose Sodium 30.000 6. Microcrystalline cellulose 300.000 7.Magnesium Stearate 4.000 Blending and Lubrication 8. Croscarmellosesodium 30.00 9. Microcrystalline cellulose 100.00 10. Magnesium Stearate6.00 Rilpivirine Part (Binder Slurry) 11. Rilpivirine Hydrochloride27.500 12. Lactose monohydrate 13.000 13. Povidone 3.250 14. Polysorbate20 0.350 15. Purified water 110 Granulation 16. Lactose monohydrate50.000 17. Crospovidone 5.000 Blending and Lubrication 18. Crospovidone3.000 19. Silicified Microcrystalline cellulose 16.800 20. Magnesiumstearate 1.100 Film Coating 21. Opadry II 85G18490 White 30.00 22.Purified water q.s Final Tablet weight 1020.00

Process:

1) Emtricitabine, Tenofovir disoproxil fumarate, piperine, Lactosemonohydrate, Microcrystalline cellulose, Croscarmellose sodium andmagnesium stearate was blended and compacted into granular mass.2) Microcrystalline cellulose, Croscarmellose sodium and magnesiumstearate was blended and compacted into granular mass.3) Polysorbate 80, Povidone and lactose was dissolved in water.4) Rilpivirine was added to the solution in obtained in step (3) to forma slurry.5) Dry mix of lactose monohydrate and crospovidone was added to theslurry obtained in step (4).6) Microcrystalline cellulose, Crospovidone and magnesium stearate wasadded to the dry blend obtained in step (5).7) The blend obtained in step (1) was compressed with the blend obtainedin step (6) to form a bilayer tablet with film coating.

Example 12

TABLE 12 Sr. No. Ingredients Qty/Tab (mg) Dry Mix 1. Emtricitabine200.000 2. Tenofovir Disoproxil Fumarate 100.000 3. Piperine 20.00 4.Lactose monohydrate 80.000 5. Croscarmellose Sodium 30.000 6.Microcrystalline cellulose 300.000 7. Magnesium Stearate 4.000 Blendingand Lubrication 8. Croscarmellose sodium 30.00 9. Microcrystallinecellulose 100.00 10. Magnesium Stearate 6.00 Total 870.00 Film Coating11. Opadry II 85G18490 White 30.00 12. Purified water q.s Final Tabletweight 900.00

Process:

1) Emtricitabine, Tenofovir disoproxil fumarate, piperine, Lactosemonohydrate, Microcrystalline cellulose, Croscarmellose sodium andmagnesium stearate were mixed and blended to form a granular mass.2) Microcrystalline cellulose, Croscarmellose sodium and magnesiumstearate were mixed and blended.3) The blend obtained in step (1) and step (2) was compressed and coatedto form tablets with film coating.

Example 13

TABLE 13 Sr. Qty/Tab No. Ingredients (mg) Dry Mix (Emtricitabine +Piperine + TDF Part) 1. Emtricitabine 200.000 2. Tenofovir DisoproxilFumarate 100.000 3. Piperine 20.00 4. Lactose monohydrate 80.000 5.Croscarmellose Sodium 30.000 6. Microcrystalline cellulose 300.000 7.Magnesium Stearate 4.000 Blending and Lubrication 8. Croscarmellosesodium 30.00 9. Microcrystalline cellulose 100.00 10. Magnesium Stearate6.00 Weight of Emtricitabine, piperine & 870.00 TDF Layer Dry Mix(Efavirenz Part) 11. Efavirenz 600.00 12. Microcrystalline cellulose202.00 13. Sodium lauryl sulphate 6.00 14. Croscarmellose sodium 48.0015. Hydroxypropyl cellulose 38.40 16. Purified water qs Blending andLubrication 17. Lactose Monohydrate 199.60 18. Magnesium Stearate 6.0019. Weight of Efavirenz layer 1100 Total weight of uncoated tablet 1970Seal Coating 20. Hypromellose 10.00 21. Isopropyl Alcohol q.s 22.Purified water q.s Total 1980.00 Film Coating 23. Opadry II 85G18490White 60.00 24. Purified water q.s Final Tablet weight 2040.00

Process:

1) Emtricitabine, Tenofovir disoproxil fumarate, piperine, Lactosemonohydrate, Microcrystalline cellulose, Croscarmellose sodium andmagnesium stearate were mixed and blended to form a granular mass.2) Microcrystalline cellulose, Croscarmellose sodium and magnesiumstearate were added to the blend obtained in step (1) and furtherblended.3) Efavirenz, microcrystalline cellulose and croscarmellose sodium wereadded to SLS followed by Hydroxypropyl cellulose to form a solution andgranulated.4) Lactose monohydrate and magnesium stearate were blended andcompressed to form a bilayer tablet having with film coating.

Example 14

TABLE 14 Sr. No. Ingredients Qty/Tab (mg) Dry Mix (Lamivudine Part) 1.Lamivudine 300.00 2. Microcrystalline cellulose 50.80 3. Croscarmellosesodium 22.50 4. Pregelatinized starch 18.00 5. Magnesium Stearate 2.50Dry Mix (Tenofovir Disoproxil Fumarate Part) 6. Tenofovir DisoproxilFumarate 100.00 7 Piperine 20.00 8. Microcrystalline cellulose 45.20 9.Croscarmellose sodium 22.50 10. Magnesium Stearate 2.50 Blending andLubrication 11. Croscarmellose sodium 30.00 12. Microcrystallinecellulose 100.00 13. Magnesium Stearate 6.00 Weight of Lamivudine,piperine & 720.00 TDF Layer Dry Mix (Efavirenz Part) 14. Efavirenz600.00 15. Microcrystalline cellulose 202.00 16. Sodium lauryl sulphate6.00 17. Croscarmellose sodium 48.00 18. Hydroxypropyl cellulose 38.4019. Purified water qs Blending and Lubrication 20. Lactose Monohydrate199.60 21. Magnesium Stearate 6.00 Weight of Efavirenz layer 1100 Totalweight of uncoated tablet 1820 Seal Coating 22. Hypromellose 10.00 23.Isopropyl Alcohol q.s 24. Purified water q.s Total 1820.00 Film Coating25. Opadry II 85G18490 White 55.00 26. Purified water q.s Final Tabletweight 1875.00

Process:

1) Lamivudine, Microcrystalline cellulose, Croscarmellose sodium,Pregelatinised starch and magnesium stearate was blended to form agranular mass.2) Tenofovir disoproxil fumarate, Piperine, Microcrystalline cellulose,Croscarmellose sodium and magnesium stearate was blended to form agranular mass.3) Microcrystalline cellulose, Croscarmellose sodium and magnesiumstearate was mixed with the blends obtained in step (1) and step (2).4) Efavirenz, microcrystalline cellulose and croscarmellose sodium wereadded to SLS followed by Hydroxypropyl cellulose to form a solution andgranulated.5) Lactose monohydrate and magnesium stearate were blended andcompressed to form a bilayer tablet having with seal coating followed byfilm coating.

In order that this invention be more fully understood, the followingpreparative and testing methods are set forth. These methods are for thepurpose of illustration only and are not to be construed as limiting thescope of the invention in any way.

Preparative and Testing Methods I) Material

Caffeine (high permeable marker), Atenolol (low permeable marker),Digoxin (known P-gp substrate), TDF (MD1431532), TAF (VRD-1063/16/187),HBSS buffer, MES hydrate, HEPES powder, Fetal bovine serum (FBS),Minimum essential medium (MEM), Lucifer yellow, Piperine (P-gpinhibitor)

Method

1) Caco-2 Cell Culture

Caco-2 cells were cultured in MEM media with 10% serum and seeded at adensity of 75000 cells per mL and cultured for 21 days in a 24-welltrans-well plate at 37° C., 5% CO₂. The monolayer integrity was checkedintermittently (Day 0-21) using Trans Epithelial Electric Resistance(TEER). Cells were treated with drugs as follows:

2) Unidirectional Assay (A-B)

Stock preparations: 10 mM stocks of all the drugs were prepared in DMSO.The test concentrations were further prepared in HBSS buffer containing10 mM MES hydrate pH 6.8 as per the plate plan. Also, HBSS buffer with10 mM HEPES with pH 7.4 was prepared.

Study Plan

Plate Setup

TABLE 15 1 2 3 A Caffeine 10 μM TDF 50 μM TAF 50 μM B (A-B) (A-B) (A-B)C Atenolol 50 μM TDF 100 μM TAF 100 μM D (A-B) (A-B) (A-B) Batch noPositive control MD1431532 VRD- 1063/16/187

Assay Protocol

0400 μL samples were added to the wells as per the plate setup to theapical side (A) prepared in 10 mM MES hydrate pH 6.8 in duplicates and800 μL HBSS with 10 mM HEPES pH 7.4 was added to all basal wells (B).Samples were collected at 60, 90 and 120 minutes from the basal side.Mass balance samples at 0 and 120 minutes were collected from the apicalside. The sample were analyzed on LCMS-MS.

4) Bidirectional Assay (A-B and B-A) to Study the Effect of Piperine(P-Gp Inhibitor) on the Permeability

Plate Plan

TABLE 16 1 2 3 4 5 6 A Digoxin Digoxine TAF 100 μM TAF 100 μM TAF 100 μMTAF 100 μM B 10 μM 10 μM (VRD- (VRD- (VRD- (VRD- A-B (A-B) +1063/16/187) 1063/16/187) + 1063/16/187) + 1063/16/187) + Piperine A-BPiperine 0.1 μM Piperine 1 μM Piperine 10 μM 10 μM A-B A-B A-B A-B CDigoxin Digoxin TAF 100 μM TAF 100 μM TAF 100 μM TAF 100 μM D 10 μM 10μM (VRD- (VRD- (VRD- (VRD- B-A (B-A) + 1063/16/187) 1063/16/187) +1063/16/187) + 1063/16/187) + Piperine B-A Piperine 0.1 μM Piperine 1 μMPiperine 10 μM 10 μM B-A B-A B-A B-A

Assay Protocol

400 μL samples were added to the wells as per the plate setup to theapical side in duplicates with 800 μL HBSS pH 7.4 in the basal wells.Samples were collected at 60, 90 and 120 minutes from the basal side.Mass balance samples at 0 and 120 minutes were collected from the apicalside.

For B-A, 800 μL of the respective dilutions were added to the basal sidein duplicates with 400 μL HBSS pH 7.4 in the apical wells. Samples werecollected at 60, 90 and 120 minutes from the apical side. Mass balancesamples at 0 and 120 minutes were collected from the basal side. Thesample were analyzed on LCMS-MS.

At the end of the experiment the monolayer integrity was checked usingLucifer yellow, and calculating the % rejection of Lucifer yellow byincubating cells with 100 μg/mL Lucifer.

5) Data Analysis:

Papp was calculated as follows:

The apparent permeability (Papp) in units per second can be calculatedby using the following equation,

Papp=(V/(T*A))*(C0/Ct)  For Single Point Method

Papp=(dQ/dt)/(A*C0)  For Multi-Point Method

% Mass balance=100−[CR120*VR+CD120*VD/C0*VD]  For Multi-Point Method

% Lucifer YellowPassage=[RFU(test)−RFU(blank)/RFU(equilibrium)−RFU(blank)]*100  ForLucifer Yellow,

Permeability Classification:

TABLE 17 Permeability Papp (nm/s) Low  <50 Moderate 50-200 High >200Efflux ratio=Papp B-A/Papp A-BEfflux ratio ≧2 indicates that the drug is a P-gp substrate

Results

Unidirectional Assay (FIG. 1)

TABLE 18 Papp (A-B) Drug Concentration (μM) nm/s Caffeine 10 921.89Atenolol 50 32.77 TDF (MD1431532) 50 65.84 100 63.54 TAF (VRD- 50 58.591063/16/187) 100 53.89

Bidirectional Assay (FIG. 2)

TABLE 19 A-B B-A Papp Papp Efflux % P-gp Sample (nm/s) (nm/s) ratioinhibition Digoxine 10 μM 19.37 204.67 10.57 0.00 Digoxine 10 μM (A-B) +96.08 196.28 2.04 80.67 Piperine 10 μM TAF 100 μM (VRD- 34.49 178.205.17 0.00 1063/16/187) TAF 100 μM (VRD- 282.66 332.71 1.18 77.221063/16/187) + Piperine 0.1 μM TAF 100 μM (VRD- 203.76 298.88 1.47 71.611063/16/187) + Piperine 1 μM TAF 100 μM (VRD- 241.43 236.81 0.98 81.021063/16/187) + Piperine 10 μM

Conclusions

The TDF and TAF are observed to be low to moderately permeable drugs.Further, TAF absorption is increased with piperine by decreasing theefflux ratio of TAF. The above data indicates that TAF is a substrate ofefflux transporter and thus its bioavailability is low. As can be seenin the data, the A-B Papp was 34.49 nm/s and its efflux ratio was 5.17.By adding piperine which is a known inhibitor of efflux transporters,the A-B, Papp increased to more than 282.66 nm/s while the efflux ratiodecreased to less than 1.18. Thus indicating addition of piperineimproves the permeability. Therefore, it can be concluded that the useof piperine decreases efflux ratio which in turn would increase itsbioavailability.

II) Material

Digoxin (known P-gp substrate), Dolutegravir (KK1406229), HBSS buffer,MES hydrate, HEPES powder, Fetal bovine serum (FBS), Minimum essentialmedium (MEM), Lucifer yellow, Piperine (P-gp inhibitor), Cobicistat(P-gp inhibitor).

Method

1) Caco-2 Cell Culture

Caco-2 cells were cultured in MEM media with 10% serum and seeded at adensity of 75000 cells per mL and cultured for 21 days in a 24-welltrans-well plate at 37° C., 5% CO₂. The monolayer integrity was checkedintermittently (Day 0-21) using Trans Epithelial Electric Resistance(TEER). Cells were treated with drugs as follows:

2) Bidirectional Assay (A-B and B-A) to Study the Effect of Piperine(P-Gp Inhibitor) on the Permeability

Plate Plan

TABLE 20 1 2 3 4 5 6 A Digoxine Dolutegravir Dolutegravir DolutegravirDolutegravir Dolutegravir B 10 μM 5 μM A-B 5 μM + 5 μM + 5 μM + 5 μM +(A-B) Piperine Piperine Verapamil Verapamil 1 μM A-B 10 μM A-B 1 μM A-B10 μM A-B C Digoxine Dolutegravir Dolutegravir Dolutegravir DolutegravirDolutegravir D 10 μM 5 μM B-A 5 μM + 5 μM + 5 μM + 5 μM + (B-A) PiperinePiperine Verapamil Verapamil 1 μM B-A 10 μM B-A 1 μM B-A 10 μM B-A

Assay Protocol

400 μL samples were added to the wells as per the plate setup to theapical side in duplicates with 800 μL HBSS pH 7.4 in the basal wells.Samples were collected at 60, 90 and 120 minutes from the basal side.Mass balance samples at 0 and 120 minutes were collected from the apicalside.

For B-A, 800 μL of the respective dilutions were added to the basal sidein duplicates with 400 μL HBSS pH 7.4 in the apical wells. Samples werecollected at 60, 90 and 120 minutes from the apical side. Mass balancesamples at 0 and 120 minutes were collected from the basal side.

The samples were analyzed on LCMS-MS. At the end of the experiment themonolayer integrity was checked using Lucifer yellow, and calculatingthe % rejection of Lucifer yellow by incubating cells with 100 μg/mLLucifer.

3) Data Analysis:

Papp was calculated as follows:

The apparent permeability (Papp) in units per second can be calculatedby using the following equation,

Papp=(V/(T*A))*(C ₀ /C _(t))  For Single Point Method

Papp=(dQ/dt)/(A*C ₀)

% Mass balance=100−[C _(R120) *V _(R) +C _(D120) *V _(D) /C ₀ *V_(D)]  For Multi-Point Method

% Lucifer YellowPassage=[RFU(test)−RFU(blank)/RFU(equilibrium)−RFU(blank)]*100  ForLucifer Yellow

Permeability Classification:

TABLE 21 Permeability Papp (nm/s) Low  <50 Moderate 50-200 High >200Efflux ratio=Papp B-A/Papp A-B

Efflux ratio ≧2 indicates that the drug is a P-gp substrate

Results

Bidirectional Assay (FIG. 3)

TABLE 22 Papp nm/s Efflux Drug A-B B-A ratio Digoxin 10 μM 44.99 288.716.41 Dolutegravir 5 μM 401.74 842.94 2.09 Dolutegravir 5 μM + Piperine 1μM 374.73 783.75 3.50 Dolutegravir 5 μM + Piperine 10 μM 328.09 809.942.46 Dolutegravir 5 μM + Verapamil 348.97 696.16 1.99 1 μM Dolutegravir5 μM + Verapamil 351.20 799.57 2.27 10 μM

Conclusions

Dolutegravir is a known P-gp substrate. Dolutegravir is a high permeabledrug and piperine does not affect the permeability of dolutegraviracross the caco-2 monolayer. Therefore, it can be concluded that the useof piperine decreases efflux ratio which in turn would increase itsbioavailability.

III) Material

Digoxin (known P-gp substrate), Darunavir (DN0011215), HBSS buffer, MEShydrate, HEPES powder, Fetal bovine serum (FBS), Minimum essentialmedium (MEM), Lucifer yellow, Piperine (P-gp inhibitor), Cobicistat(P-gp inhibitor)

Method

1.) Caco-2 Cell Culture

Caco-2 cells were cultured in MEM media with 10% serum and seeded at adensity of 75000 cells per mL and cultured for 21 days in a 24-welltrans-well plate at 37° C., 5% CO₂. The monolayer integrity was checkedintermittently (Day 0-21) using Trans Epithelial Electric Resistance(TEER). Cells were treated with drugs as follows:

2.) Bidirectional Assay (A-B and B-A) to Study the Effect of Piperine(P-Gp Inhibitor) on the Permeability

Plate Plan

TABLE 23 1 2 3 4 5 6 A Digoxine Darunavir Darunavir Darunavir DarunavirDarunavir B 10 μM 40 μM A-B 40 μM + 40 μM + 40 μM + 40 μM + (A-B)Piperine Piperine Cobicistat Cobicistat 1 μM A-B 10 μM A-B 10 μM A-B 100μM A-B C Digoxine Darunavir Darunavir Darunavir Darunavir Darunavir D 10μM 40 μM B-A 40 μM + 40 μM + 40 μM + 40 μM + (B-A) Piperine PiperineCobicistat Cobicistat 1 μM B-A 10 μM B-A 10 μM B-A 100 μM B-A

Assay Protocol

400 μL samples were added to the wells as per the plate setup to theapical side in duplicates with 800 μL HBSS pH 7.4 in the basal wells.Samples were collected at 60, 90 and 120 minutes from the basal side.Mass balance samples at 0 and 120 minutes were collected from the apicalside.

For B-A, 800 μL of the respective dilutions were added to the basal sidein duplicates with 400 μL HBSS pH 7.4 in the apical wells. Samples werecollected at 60, 90 and 120 minutes from the apical side. Mass balancesamples at 0 and 120 minutes were collected from the basal side.

The sample were analyzed on LCMS-MS. At the end of the experiment, themonolayer integrity was checked using and Lucifer yellow, calculatingthe % rejection of Lucifer yellow by incubating cells with 100 μg/mLLucifer.

3.) Data Analysis:

Papp was calculated as follows:

The apparent permeability (Papp) in units per second can be calculatedby using the following equation,

Papp=(V/(T*A))*(C0/Ct)  For Single Point Method

Papp=(dQ/dt)/(A*C0)

% Mass balance=100−[CR120*VR+CD120*VD/C0*VD]  For Multi-Point Method

% Lucifer YellowPassage=[RFU(test)−RFU(blank)/RFU(equilibrium)−RFU(blank)]*100  ForLucifer Yellow,

Permeability Classification:

TABLE 24 Permeability Papp (nm/s) Low  <50 Moderate 50-200 High >200Efflux ratio=Papp B-A/Papp A-BEfflux ratio ≧2 indicates that the drug is a P-gp substrate

Results

Bidirectional Assay (FIG. 4)

TABLE 25 Papp nm/s Efflux Drug A-B B-A ratio Digoxin 10 μM 11.17 142.1312.73 Darunavir 40 μM 88.35 217.39 2.46 Darunavir 40 μM + Piperine 1 μM103.08 360.83 3.50 Darunavir 40 μM + Piperine 10 μM 202.37 236.27 1.17Darunavir 40 μM + Cobicistat 341.95 222.38 0.65 10 μM Darunavir 40 μM +Cobicistat 250.50 121.57 0.49 100 μM

Conclusions

Darunavir is a known P-gp substrate. Absorption of Darunavir isincreased with piperine by decreasing the efflux ratio of TAF.Therefore, it can be concluded that the use of piperine decreases effluxratio which in turn would increase its bioavailability.

IV) Material

Digoxin (known P-gp substrate), TDF, HBSS buffer, MES hydrate, HEPESpowder, Fetal bovine serum (FBS), Minimum essential medium (MEM),Lucifer yellow, Piperine (P-gp inhibitor), Cobicistat (P-gp inhibitor),Tetrahydropiperine (P-gp inhibitor)

Method

1.) Caco-2 Cell Culture

Caco-2 cells were cultured in MEM media with 10% serum and seeded at adensity of 75000 cells per mL and cultured for 21 days in a 24-welltrans-well plate at 37° C., 5% CO₂. The monolayer integrity was checkedintermittently (Day 0-21) using Trans Epithelial Electric Resistance(TEER). Cells were treated with drugs as follows:

2.) Bidirectional Assay (A-B and B-A) to Study the Effect of Piperine(P-Gp Inhibitor) and Tetrahydropiperine on the Permeability

Plate Plan

TABLE 26 1 2 3 4 5 A Digoxine TDF TDF TDF 100 μM TDF 100 μM B 10 μM (A-200 μM 100 μM (A-B) + (A-B) + B) (A-B) (A-B) Piperine 10 μM THpiperine10 μM C Digoxine TDF TDF TDF 100 μM TDF 100 μM 10 μM 200 μM 100 μM(B-A) + (B-A) + (B-A) (B-A) (B-A) Piperine 10 μM Thpiperine 10 μM

Assay Protocol

400 μL samples were added to the wells as per the plate setup to theapical side in duplicates with 800 μL HBSS pH 7.4 in the basal wells.Samples were collected at 60, 90 and 120 minutes from the basal side.Mass balance samples at 0 and 120 minutes were collected from the apicalside.

For B-A, 800 μL of the respective dilutions were added to the basal sidein duplicates with 400 μL HBSS pH 7.4 in the apical wells. Samples werecollected at 60, 90 and 120 minutes from the apical side. Mass balancesamples at 0 and 120 minutes were collected from the basal side.

The sample were analyzed on LCMS-MS. At the end of the experiment themonolayer integrity was checked using and Lucifer yellow, calculatingthe % rejection of Lucifer yellow by incubating cells with 100 μg/mLLucifer.

3.) Data Analysis:

Papp was calculated as follows:

The apparent permeability (Papp) in units per second can be calculatedby using the following equation,

Papp=(V/(T*A))*(C0/Ct)  For Single Point Method

Papp=(dQ/dt)/(A*C0)

% Mass balance=100−[CR120*VR+CD120*VD/C0*VD]  For Multi-Point Method:

For Lucifer yellow, % Lucifer YellowPassage=[RFU(test)−RFU(blank)/RFU(equilibrium)−RFU(blank)]*100

Permeability Classification:

TABLE 27 Permeability Papp (nm/s) Low  <50 Moderate 50-200 High >200Efflux ratio=Papp B-A/Papp A-BEfflux ratio ≧2 indicates that the drug is a P-gp substrate

Results:

Bidirectional Assay (FIG. 5)

TABLE 28 Papp nm/s Efflux Drug A-B B-A ratio Digoxin 10 μM 8.16 154.7418.95 TDF 200 μM 37.67 142.76 3.79 TDF 100 μM 25.51 152.23 5.97 TDF 100μM + Piperine 10 μM 43.37 78.22 1.80 TDF 100 μM + Tetrahydro Piperine 10μM 50.22 90.77 1.81

Conclusions

TDF is a known P-gp substrate. Absorption of TDF is increased withpiperine by decreasing the efflux ratio. Further, absorption of TDF isincreased with tetrahydropiperine by decreasing the efflux ratio.Comparable improvement in permeability of TDF was seen by both Piperineand tetrahydropiperine. Therefore, it can be concluded that the use ofpiperine and tetrahydropiperine decreases efflux ratio which in turnwould increase its bioavailability.

Animal Study

In Vivo Rat PK Study

The objective of this non-GLP study was to determine thepharmacokinetics of the TDF alone and in combinations with piperine indifferent groups of male Wistar rats after single dose intravenousadministration and oral administration. This study was performed withapproval from the Institutional Animal Ethics Committee (IAEC) inaccordance with the requirement of Committee for the Purpose of Controland Supervision of Experiments on Animals (CPCSEA), India.

Study Design

The study was conducted using six male Wistar rats in each group asshown in Table 30 below.

TABLE 29 Formu- Dose Concen- Test lation Dose vol tration Group compoundvehicle Route mg/kg mL/kg (mg/mL) 1 TDF Normal IV 7.75 5 1.55 saline 2TDF 0.5% PO 31 5 6.2 Sodium Carboxy Methyl Cellulose in double distilled(DS) water 3 TDF 0.5% PO 15.5 5 3.1 Sodium Carboxy Methyl Cellulose inDS water 4 TDF + 0.5% PO 31 + 2  5 6.2 + 0.4 Piperine Sodium CarboxyMethyl Cellulose in DS water 5 TDF + 0.5% Sodium PO 15.5 + 2   5 3.1 +0.4 Piperine Carboxy Methyl Cellulose in DS water

Formulation Preparation

The solution formulations were prepared as follows:

Intravenous Route:

Required quantity of Compound B (TDF) (23.03 mg) was weighed and to this10.94 mL of vehicle (Normal saline) was added, vortexed and sonicatedfor 2 minutes to make a clear formulation.

Per Oral Route:

For Group 2:

Required quantity of Compound B (79.37 mg) was weighed and to this 9.42mL of vehicle (Na Carboxy Methylcellulose) was added, vortexed andsonicated for 2 minutes to make formulation of 6.2 mg/mL concentration.

For Group 3:

Required quantity of Compound B (46.14 mg) was weighed and to this 10.95mL of vehicle (Na Carboxy Methylcellulose) added, vortexed and sonicatedfor 2 minutes to make a uniform formulation.

For Group 4:

Required quantity of Compound B (79.56 mg) was weighed and to this 4.72mL of vehicle (Na Carboxy Methylcellulose) was added, vortexed andsonicated for 2 minutes to make a clear formulation. Required quantityof Compound B 1 (piperine) (18.95 mg) was weighed and to this 22.71 mLof vehicle (Na Carboxy Methylcellulose) was added, vortexed andsonicated for 2 minutes to make a clear formulation. An equal volume(4.72 mL) of each formulation was mixed in separate vials to get 5mL/kg.

For Group 5:

Required quantity of Compound B (47.20 mg) and Compound BI (18.95 mg)were weighed and to this 5.61 mL of vehicle (Na Carboxy Methylcellulose)was added, vortexed and sonicated for 2 minutes to make a clearformulation. An equal volume (5.61 mL) of each formulation was mixed inseparate vials to get 5 mLlkg.

Bloanalysis

Bioanalysis was performed using fit-for-purpose LC-MS/MS method for thequantification of TDF and PMPA in rat plasma samples. The calibrationcurve (CC) for the method consisted of nine non-zero calibrationstandards along with a double blank and zero standard samples.

Pharmacokinetic Analysis

Plasma pharmacokinetic parameters were calculated using thenon-compartmental analysis tool of Phoenix software (Version 6.3) andwere determined from individual animals in each group. The peak plasmaconcentration (Cmax), time to achieve peak plasma concentration (Tmax),the area under the plasma concentration-time curve (AUC0-t and AUCinf),AUC Extra (%), elimination half-life (T½), clearance (CL), volume ofdistribution Vd (Lkg) and Mean residence time (MRT) were calculated fromthe intravenous group. The peak plasma concentration (Cmax), time toachieve peak plasma concentration (Tmax), AUC0-t and AUCinf, AUC Extra(%), Mean residence time (MRT) and absolute oral bioavailability (F)were calculated from the oral group.

Results

The plasma concentration-time data and plasma pharmacokinetic parametersof PMPA following intravenous and oral administration of TDF in maleWistar rats are presented in the Table 31.

TABLE 30 Route/Dose T_(max) C_(max) AUC_(0-t) AUC_(0-inf) (mg/kg) (h)(ng/mL) (h * ng/mL) (h * ng/mL) F (%) IV (7.75) G1 0.08 ± 0.00 2425.44 ±677.91  900.66 ± 213.26 1023.37 ± 217.13 NA PO (31) G2 0.71 ± 0.33294.14 ± 140.55 1284.52 ± 392.13  1407.62 ± 402.73 34.39 ± 6.07 PO(15.5) G3 0.71 ± 0.70 283.43 ± 108.44 846.94 ± 128.87  904.96 ± 111.7644.22 ± 1.91 PO (B = 31 + 0.58 ± 0.20 462.52 ± 68.80  1487.98 ± 174.80 1586.43 ± 164.81 38.76 ± 4.82 B1 = 2) G4 PO (B = 15.5 + 0.50 ± 0.00340.07 ± 118.97 955.15 ± 250.07 1074.19 ± 307.58  52.48 ± 13.48 B1 = 2)G5

FIGS. 6 and 7 show Plasma concentration of tenofovir for TDF 300 mg andTDF 300 mg+Piperine 20 mg at different time points.

TABLE 31 Cmax, Tmax and AUC values of different combinations 300 mg 150mg TDF + TDF + 300 mg 20 mg 150 mg 20 mg TDF Piperine TDF Piperine Cmax462.26 727.23 444.96 534.59 (nM) Tmax (h) 0.71 0.58 0.71 0.5 AUC 1407.621586.53 904.96 1074.19

FIG. 8 shows time dependent plasma concentration of tenofovir for 300 mgTDF, 300 mg TDF+20 mg piperine and 150 mg TDF+20 mg piperine

Conclusions

The rat PK study clearly indicates that the peak plasma concentration oftenofovir significantly increased when TDF is administered incombination with piperine. The results demonstrate a significantbioavailability enhancement when TDF at 150 mg is administered with 20mg piperine (52.48±13.48) as compared to TDF 300 mg alone (34.39±6.07).

The compositions and methods of the appended claims are not limited inscope by the specific compositions and methods described herein, whichare intended as illustrations of a few aspects of the claims and anycompositions and methods that are functionally equivalent are intendedto fall within the scope of the claims. Various modifications of thecompositions and methods in addition to those shown and described hereinare intended to fall within the scope of the appended claims. Further,while only certain representative compositions and method stepsdisclosed herein are specifically described, other combinations of thecompositions and method steps also are intended to fall within the scopeof the appended claims, even if not specifically recited. Thus, acombination of steps, elements, components, or constituents may beexplicitly mentioned herein or less, however, other combinations ofsteps, elements, components, and constituents are included, even thoughnot explicitly stated. The term “comprising” and variations thereof asused herein is used synonymously with the term “including” andvariations thereof and are open, non-limiting terms. Although the terms“comprising” and “including” have been used herein to describe variousembodiments, the terms “consisting essentially of” and “consisting of”can be used in place of “comprising” and “including” to provide for morespecific embodiments of the invention and are also disclosed. Other thanin the examples, or where otherwise noted, all numbers expressingquantities of ingredients, reaction conditions, and so forth used in thespecification and claims are to be understood at the very least, and notas an attempt to limit the application of the doctrine of equivalents tothe scope of the claims, to be construed in light of the number ofsignificant digits and ordinary rounding approaches.

All patent and non-patent publications cited in this disclosure areincorporated herein in to the extent as if each of those patent andnon-patent publications was incorporated herein by reference in itsentirety. Further, even though the disclosure herein has been describedwith reference to particular examples and embodiments, it is to beunderstood that these examples and embodiments are merely illustrativeof the principles and applications of the present disclosure. It istherefore to be understood that numerous modifications may be made tothe illustrative embodiments and that other arrangements may be devisedwithout departing from the spirit and scope of the present disclosure asdefined by the following claims.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to various embodimentsdescribed herein without departing from the spirit or scope of theteachings herein. Thus, it is intended that various embodiments coverother modifications and variations of various embodiments within thescope of the present teachings.

What is claimed:
 1. An oral or injectable pharmaceutical compositioncomprising a therapeutically effective amount of at least oneanti-retroviral drug and a therapeutically effective amount of at leastone pharmacokinetic booster or enhancer or derivative thereof.
 2. Theoral or injectable pharmaceutical composition of claim 1, wherein (i)the at least one pharmacokinetic booster or enhancer or derivativethereof reduces a dosing frequency of the at least one anti-retroviraldrug that is administered to a patient; and (ii) the at least onepharmacokinetic booster or enhancer or derivative thereof increases thebioavailability of the at least one anti-retroviral drug from about 10%to about 70%.
 3. The oral or injectable pharmaceutical composition ofclaim 1, wherein (i) the at least one anti-retroviral drug comprises anucleoside reverse transcriptase inhibitor (NRTI), non-nucleosidereverse transcriptase inhibitor (NNRTI), nucleotide analogreverse-transcriptase inhibitor, protease inhibitor (PI), integraseinhibitor, fusion inhibitor, CCR5 inhibitor, monoclonal antibody,glycoprotein inhibitor or combinations thereof; and (ii) the at leastone pharmacokinetic booster comprises piperine, tetrahydropiperine,cis-piperine, trans-piperine, cis-trans piperine, trans,cis-piperine,cis,cis-piperine, trans,trans-piperine or a combination thereof.
 4. Theoral or injectable pharmaceutical composition of claim 3, wherein the atleast one anti-retroviral drug comprises a nucleoside reversetranscriptase inhibitor (NRTI) comprising lamivudine, abacavir,zidovudine, emtricitabine, didanosine, stavudine, entecavir,apricitabine, censavudine, zalcitabine, dexelvucitabine, amdoxovir,elvucitabine, festinavir, racivir, stampidine or a combination thereof;a non-nucleoside reverse transcriptase inhibitor (NNRTI) comprisinglersivirine, rilpivirine, efavirenz, etravirine, doravirine, dapivirineor a combination thereof; a nucleotide analog reverse-transcriptaseinhibitor comprising tenofovir alafenamide fumarate, tenofovirdisoproxil fumarate, adefovir or a combination thereof; a proteaseinhibitor (PI) comprising lopinavir, ritonavir, saquinavir, nelfinavir,amprenavir, indinavir, nelfinavir, atazanavir, lasinavir, palinavir,tirpranavir, fosamprenavir, darunavir, tipranavir or a combinationthereof; an integrase inhibitor comprising dolutegravir, elvitegravir,raltegravir, bictegravir, cabotegravir or a combination thereof; afusion inhibitor comprising maraviroc, enfuvirtide, griffithsin,aplaviroc, vicriviroc, plerixafor, fostemsavir, albuvirtide or acombination thereof; a CCR5 inhibitor comprising aplaviroc, vicriviroc,maraviroc, cenicriviroc or a combination thereof; a monoclonal antibodycomprising ibalizumab; a glycoprotein inhibitor comprising sifuvirtide;or combinations thereof.
 5. The oral or injectable pharmaceuticalcomposition of claim 1, wherein the ratio of the at least oneanti-retroviral drug to the at least one pharmacokinetic booster orenhancer or derivative thereof is from about 100:1 to about 1:1 byweight.
 6. The oral or injectable pharmaceutical composition of claim 4,wherein the tenofovir disoproxil fumarate in the composition is fromabout 1 mg to about 300 mg.
 7. The oral or injectable pharmaceuticalcomposition of claim 4, wherein the tenofovir alafenamide fumarate inthe composition is from about 1 to about 25 mg.
 8. The oral orinjectable pharmaceutical composition of claim 4, wherein thedolutegravir in the composition is from about 1 mg to about 50 mg. 9.The oral or injectable pharmaceutical composition of claim 4, whereinthe darunavir in the composition is from about 1 mg to about 800 mg. 10.The oral or injectable pharmaceutical composition of claim 4, wherein(i) the elvitegravir in the composition is from about 1 mg to about 150mg; and (ii) the raltegravir in the composition is from about 1 mg toabout 400 mg.
 11. The oral or injectable pharmaceutical composition ofclaim 3, wherein the piperine is in the composition from about 0.5 mg toabout 400 mg.
 12. An oral or injectable pharmaceutical compositioncomprising a therapeutically effective amount of at least oneanti-retroviral drug; a therapeutically effective amount of at least onepharmacokinetic booster or enhancer or derivative thereof; and one ormore pharmaceutically acceptable excipients comprising carriers,diluents, fillers, binders, lubricants, glidants, disintegrants, bulkingagents, flavorants or any combination thereof.
 13. The oral orinjectable pharmaceutical composition of claim 12, wherein the oralcomposition is in the form of a tablet, mini-tablet, granules,sprinkles, capsules, sachets, powders, pellets, and the injectablecomposition is in the form of a solution, suspension, emulsion,lyophilized powder or in the form of a kit.
 14. The oral or injectablepharmaceutical composition of claim 12, wherein the oral or injectablepharmaceutical composition is for use in the treatment or prophylaxis ofdiseases caused by retroviruses.
 15. The oral or injectablepharmaceutical composition of claim 12, wherein the oral or injectablepharmaceutical composition is for use in the treatment of diseasescaused by hepatitis B viruses.
 16. A method of treating diseases causedby retroviruses or hepatitis B viruses in a patient in need of suchtreatment, the method comprising: administering a pharmaceuticalcomposition comprising (i) a therapeutically effective amount of atleast one anti-retroviral drug or an antiviral drug; (ii) atherapeutically effective amount of at least one pharmacokinetic boosteror enhancer or derivative thereof; and (iii) one or morepharmaceutically acceptable excipients comprising carriers, diluents,fillers, binders, lubricants, glidants, disintegrants, bulking agents,flavourants or any combination thereof.
 17. The method according toclaim 16, wherein the diseases caused by retroviruses comprises acquiredimmune deficiency syndrome or an HIV infection.
 18. A method of making apharmaceutical composition that enhances the bioavailability of ananti-retroviral drug, the method comprising: mixing a therapeuticallyeffective amount of at least one anti-retroviral drug and atherapeutically effective amount of at least one pharmacokinetic boosteror enhancer or derivative thereof with one or more pharmaceuticallyacceptable excipients to make the pharmaceutical composition.
 19. Themethod according to claim 18, wherein the at least one anti-retroviraldrug comprises a nucleoside reverse transcriptase inhibitor (NRTI)comprising lamivudine, abacavir, zidovudine, emtricitabine, didanosine,stavudine, entecavir, apricitabine, censavudine, zalcitabine,dexelvucitabine, amdoxovir, elvucitabine, festinavir, racivir,stampidine or a combination thereof; a non-nucleoside reversetranscriptase inhibitor (NNRTI) comprising lersivirine, rilpivirine,efavirenz, etravirine, doravirine, dapivirine or a combination thereof;a nucleotide analog reverse-transcriptase inhibitor comprising tenofoviralafenamide fumarate, tenofovir disoproxil fumarate, adefovir or acombination thereof; a protease inhibitor (PI) comprising lopinavir,ritonavir, saquinavir, nelfinavir, amprenavir, indinavir, nelfinavir,atazanavir, lasinavir, palinavir, tirpranavir, fosamprenavir, darunavir,tipranavir or a combination thereof; an integrase inhibitor comprisingdolutegravir, elvitegravir, raltegravir, bictegravir, cabotegravir or acombination thereof; a fusion inhibitor comprising maraviroc,enfuvirtide, griffithsin, aplaviroc, vicriviroc, plerixafor,fostemsavir, albuvirtide or a combination thereof; a CCR5 inhibitorcomprising aplaviroc, vicriviroc, maraviroc, cenicriviroc or acombination thereof; a monoclonal antibody comprising ibalizumab; aglycoprotein inhibitor comprising sifuvirtide; or combinations thereof.20. The method according to claim 18, wherein the at least onepharmacokinetic booster or enhancer or derivative thereof comprisespiperine, tetrahydropiperine, cis-piperine, trans-piperine, cis-transpiperine, trans,cis-piperine, cis,cis-piperine, trans,trans-piperine ora combination thereof.
 21. A kit for treating disease caused byretroviruses or hepatitis B viruses, the kit comprising atherapeutically effective amount of at least one anti-retroviral drugand a therapeutically effective amount of at least one pharmacokineticbooster or enhancer or derivative thereof, wherein the at least oneanti-retroviral drug is in a separate composition from the at least onepharmacokinetic booster or enhancer or derivative thereof.
 22. A methodof enhancing the bioavailability of an oral anti-retroviral drug, themethod comprising: providing a therapeutically effective amount of atleast one anti-retroviral drug and providing a therapeutically effectiveamount of at least one pharmacokinetic booster or enhancer or derivativethereof.
 23. The method of claim 22, wherein (i) the at least oneanti-retroviral drug is in a first composition and the at least onepharmacokinetic booster or enhancer or derivative thereof is in a secondcomposition; or (ii) the at least one anti-retroviral drug and the atleast one pharmacokinetic booster or enhancer or derivative thereof iscombined in one composition.